Function of His185 in Aquifex aeolicus 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase

Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn2+ or Fe2+ in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd2+. The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe2+, but not for Zn2+. However, maximal activity is again achieved by substitution of the endogenous metal with Cd2+. We have determined the X-ray structures of the Cd2+ H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd2+ bound, suggesting that coordination by His185 is not essential to retain Cd2+ in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd2+ with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A. aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion.